目的 探讨苦杏仁-苦参（Armeniacae Semen Amarum-Sophorae Flavescentis Radix）对博来霉素诱导的特发性肺纤维化（idiopathic pulmonary fibrosis，IPF）大鼠的保护作用，并探讨其作用机制。方法 SD大鼠随机分为对照组、模型组、吡非尼酮（200 mg/kg）组和苦杏仁-苦参低、中、高剂量（1、2、4 g/kg）组，采用一次性气管滴注硫酸博来霉素（5 mg/kg）的方式建立IPF大鼠模型，给予药物干预后，采用苏木素-伊红（hematoxylin-eosin，HE）和Masson染色观察肺组织病理学改变；ELISA检测血清及肺泡灌洗液中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、羟脯氨酸（hydroxyproline，Hyp）水平；TUNEL染色检测肺组织细胞凋亡情况；免疫荧光检测肺组织中α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、Ⅰ型胶原蛋白（collagen type Ⅰ，collagen-Ⅰ）、纤连蛋白-1（fibronectin-1，FN-1）的表达；Western blotting检测肺组织p-p38、磷酸化细胞外信号调节激酶（phosphorylated extracellular signal-regulated kinase，p-ERK）、磷酸化c-Jun氨基末端激酶（phosphorylated c-Jun N-terminal kinase，p-JNK）、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）表达。采用转化生长因子-β1（transforming growth factor-β1，TGF-β1）诱导人肺成纤维细胞（MRC-5）建立体外模型，给予苦杏仁-苦参含药血清干预后，采用CCK-8法检测细胞增殖活力；TUNEL染色检测细胞凋亡情况；免疫荧光检测α-SMA、collagen-Ⅰ、FN-1的表达；Western blotting检测p-RAF、磷酸化丝裂原活化蛋白激酶激酶（phosphorylated mitogen-activated protein kinase kinase，p-MEK）、p-ERK、细胞周期蛋白D1（Cyclin D1）、增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、Bax、Bcl-2蛋白表达。结果 体内实验中，与对照组比较，模型组大鼠体质量降低（P＜0.01），肺组织干湿质量比和肺脏指数升高（P＜0.01），肺组织出现明显炎性浸润、肺泡间隙增厚及水肿，肺组织细胞凋亡率降低（P＜0.01），血清及肺泡灌洗液中炎症因子水平升高（P＜0.01），肺组织中α-SMA、collagen-Ⅰ、FN-1、p-p38、p-ERK、p-JNK、Bcl-2蛋白表达显著上调（P＜0.01），Bax蛋白表达显著下调（P＜0.01）；与模型组比较，各给药组大鼠体质量升高（P＜0.01），肺组织干湿质量比和肺脏指数降低（P＜0.01），肺组织病理变化得到明显改善，肺组织细胞凋亡率升高（P＜0.01），血清及肺泡灌洗液中炎症因子水平降低（P＜0.05、0.01），肺组织中α-SMA、collagen-Ⅰ、FN-1、p-p38、p-ERK、p-JNK、Bcl-2蛋白表达显著下调（P＜0.01），Bax蛋白表达显著上调（P＜0.01）。体外实验中，与对照组比较，模型组细胞增殖活性升高（P＜0.01），细胞凋亡率降低（P＜0.01），α-SMA、collagen-Ⅰ、FN-1、p-RAF、p-MEK、p-ERK、Cyclin D1、PCNA、Bcl-2蛋白表达升高（P＜0.01），Bax蛋白表达降低（P＜0.01）；与模型组比较，杏仁-苦参含药血清以及RAF抑制剂GW5074干预后细胞增殖活性降低（P＜0.05、0.01），细胞凋亡率升高（P＜0.01），α-SMA、collagen-Ⅰ、FN-1、p-RAF、p-MEK、p-ERK、Cyclin D1、PCNA、Bcl-2蛋白表达降低（P＜0.05、0.01），Bax蛋白表达升高（P＜0.05、0.01）。结论 苦杏仁-苦参通过调控RAF/MEK/ERK信号通路，抑制炎症反应、抑制细胞增殖以及促进细胞凋亡，从而发挥抗IPF的作用。

Objective To explore the protective effect of herb pair Kuxingren (Armeniacae Semen Amarum)-Kushen (Sophorae Flavescentis Radix) on bleomycin-induced idiopathic pulmonary fibrosis (IPF) in rats and investigate its mechanism. Methods SD rats were randomly divided into control group, model group, pirfenidone (200 mg/kg) group, Armeniacae Semen Amarum-Sophorae Flavescentis Radix low-, medium-and high-dose (1, 2, 4 g/kg) groups. The IPF rat model was established by one-time tracheal instillation of bleomycin sulfate (5 mg/kg). After drug intervention, the pathological changes in lung tissue were observed using hematoxylin-eosin (HE) and Masson staining. ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and hydroxyproline (Hyp) in serum and bronchoalveolar lavage fluid. TUNEL staining was used to detect cell apoptosis in lung tissue. The expressions of α-smooth muscle actin (α-SMA), collagen type I (collagen-I) and fibronectin-1 (FN-1) in lung tissue were detected by immunofluorescence. Western blotting was used to detect the expressions of p-p38, phosphorylated extracellular signal regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in lung tissue. Transforming growth factor-β1 (TGF-β1) was used to induce human lung fibroblasts (MRC-5) in vitro model. After intervention with Armeniacae Semen Amarum-Sophorae Flavescentis Radix containing serum, cell proliferation activity was detected using CCK-8 method. TUNEL staining was used to detect cell apoptosis. The expressions of α-SMA, collagen-I and FN-1 were detected by immunofluorescence. Western blotting was used to detect the expressions of p-RAF, phosphorylated mitogen activated protein kinase (p-MEK), p-ERK, Cyclin D1, proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 proteins. Results In the in vivo experiment, compared with control group, the body weight of rats in model group was decreased (P < 0.01), the dry wet mass ratio and lung index of lung tissue were increased (P < 0.01), the lung tissue showed significant inflammatory infiltration, thickening and edema of alveolar space, and the cell apoptosis rate of lung tissue was decreased (P < 0.01), the levels of inflammatory factors in serum and bronchoalveolar lavage fluid were increased (P < 0.01), the protein expressions of α-SMA, collagen-I, FN-1, p-p38, p-ERK, p-JNK and Bcl-2 in lung tissue were significantly up-regulated (P < 0.01), while the protein expression of Bax was significantly down-regulated (P < 0.01). Compared with model group, the body weight of rats in each treatment group were increased (P < 0.01), the dry wet mass ratio and lung index of lung tissue were decreased (P < 0.01), the pathological changes of lung tissue were significantly improved, the cell apoptosis rate of lung tissue was increased (P < 0.01), the levels of inflammatory factors in serum and bronchoalveolar lavage fluid were decreased (P < 0.05, 0.01), the expressions of α-SMA, collagen-I, FN-1, p-p38, p-ERK, p-JNK and Bcl-2 proteins in lung tissue were significantly down-regulated (P < 0.01), and the expression of Bax protein was significantly up-regulated (P < 0.01). In the in vitro experiments, compared with control group, cell proliferation activity in model group was increased (P < 0.01), cell apoptosis rate was decreased (P < 0.01), α-SMA, collagen-Ⅰ, FN-1, p-RAF, p-MEK, p-ERK, Cyclin D1, PCNA, Bcl-2 protein expressions were increased (P < 0.01), and Bax protein expression was decreased (P < 0.01). Compared with model group, Armeniacae Semen Amarum-Sophorae Flavescentis Radix containing serum and RAF inhibitor GW5074 intervention resulted in a decrease in cell proliferation activity (P < 0.05, 0.01), an increase in cell apoptosis rate (P < 0.01), a decrease in the expressions of α-SMA, collagen-I, FN-1, p-RAF, p-MEK, p-ERK, Cyclin D1, PCNA, Bcl-2 proteins (P < 0.05, 0.01), and an increase in Bax protein expression (P < 0.05, 0.01). Conclusion Armeniacae Semen Amarum-Sophorae Flavescentis Radix exerts an anti-IPF effect by regulating RAF/MEK/ERK signaling pathway, inhibiting inflammatory response, suppressing cell proliferation and promoting cell apoptosis.

R285.5

国家自然科学基金区域创新发展联合基金项目（U20A20404）；宁夏自然科学基金项目（2023AAC03212）；国家中医药管理局高水平中医药重点学科温病学学科建设项目（zyyzdxk-2023209）；2022年全国名老中医药专家传承工作室建设项目（国中医药人教函［2022］75号）